1. Field of the Invention
The present invention relates to a novel serum-free tissue culture medium useful for cultivation of a virus producer cell, in particular a retrovirus producer cell required for production of a recombinant retrovirus vector, which is used for studies of gene therapy or clinical gene therapy, and a method for producing a virus, in particular a recombinant retrovirus vector, using the medium.
2. Description of Related Art
Gene therapies in which virus vectors are used have been developed and many clinical tests have been carried out aiming at treatment of congenital genetic diseases as well as cancers and infectious diseases. In particular, a large number of trials have been conducted for gene therapy utilizing a retrovirus vector or a adenovirus vector.
Examples of DNA vectors used for production of recombinant retrovirus vectors which are used for integrating genes of interest include MFG and LXSN (GenBank Accession No. M28248) in which genes for virus particle structural proteins (gag, pol, env) are eliminated from the wild-type Moloney murine leukemia virus (MoMLV)) genome. Other vectors having further modification have been used in clinical tests for human subjects.
A recombinant retrovirus vector is produced by cultivating a producer cell which is derived from transfection of a DNA vector inserted with a gene of interest into a packaging cell (Psi-Crip, GP+E86, GP+envAm12, PG13, etc.), and collecting a supernatant which contains the virus vector of interest. A producer cell clone that stably produces a retrovirus vector for stable expression of a gene of interest may be selected, for example, from infected cells obtained by further infecting a packaging cell using the supernatant. Through such steps, a master cell bank (MCB) and a working cell bank (WCB) are prepared, and a recombinant retrovirus vector for gene therapy is stably produced.
Cultivation of a retrovirus producer cell is very important for stable retrovirus production. Usually, a retrovirus producer cell is cultivated in a serum-containing medium, and a virus-containing supernatant is collected from the culture. A case of successful cultivation under serum-free conditions has been reported. In this case, a cell capable of growing under serum-free conditions is selected in a step called adaptation in which the serum concentration in the medium is gradually decreased. However, it is generally very difficult to produce a retrovirus using a serum-free medium (Mol. Biotechnol., 15:249-257 (2000)).